Evidence in pre-clinical and clinical settings suggests the involvement of chemokines in metastatic tumor cell disease spreading. We have recently provided data validating the involvement of the CX3CR1/Fractalkine axis in breast and prostate cancer metastasis. We have discovered a potent in vivo efficacious drug-like small molecule using in vitro chemotaxis and functional assays to provide pharmacological proof of concept that modulation of the CX3CR1/ Fractalkine axis with a small molecule CX3CR1 antagonist may have therapeutic benefit in the treatment of advanced breast and prostate cancer. Specific aims of this proposal are: Aim 1: To provide an improved drug-like CX3CR1 antagonist. The lead compound, JMS-17-2, will be optimized using medicinal chemistry approaches; maintaining potency, improving selectivity, and optimizing in vitro pharmacokinetic properties and the resulting optimized lead from this aim will be evaluated for in vivo pharmacokinetics in mice and used to determine the therapeutic potential of a CX3CR1 antagonist in a pre- clinical animal model of metastasis. Aim 2: To establish a role for CX3CR1 in supporting the bone tissue colonization of cancer cells after their homing to the skeleton. We will transduce MDA-MB-436 breast cancer cells with doxycycline inducible CX3CR1-expression lentiviral particles, sort by FACS to enrich for high-expressing cells, confirm CX3CR1 conditional expression in vivo, and use these cells to determine the importance of CX3CR1 on metastatic progression in vivo. Expression of CX3CR1 will be controlled during progression by doxycycline treatment. The ability of our lead compound to inhibit metastatic progression in this model will also be evaluated. An inhibitor of metastatic dissemination will advance clinical practice and improve therapy by directly addressing tumor spreading, the direct cause of mortality in patients with advanced disease. Synergy of a CX3CR1 antagonist and a chemotherapeutic is expected due to preventing CTCs from entering the sanctuary of the bone marrow. Evidence shows that CX3CR1 antagonism extends to oncology indications such as breast adenocarcinoma, prostate, epithelial ovarian carcinoma, pancreatic cancer, and glioma. Thus, it is envisioned that an efficacious drug to treat metastatic progression would be widely applicable and have a significant impact on the morbidity and mortality associated with metastatic disease.